1. Field of the Invention
This invention provides a method of making antibody- or antibody fragment-targeted immunoliposomes and antibody- or antibody fragment-targeted polymers useful for the systemic delivery of molecules to treat diseases. The liposome and polymer complexes are useful for carrying out delivery of small molecules, as well as targeted gene delivery and efficient gene expression after systemic administration. The specificity of the delivery system is derived from the targeting antibodies or antibody fragments.
2. Related Art
The ideal therapeutic for cancer would be one that selectively targets a cellular pathway responsible for the tumor phenotype and would be nontoxic to normal cells. To date, the ideal therapeutic remains just that—an ideal. While cancer treatments involving gene therapy have substantial promise, there are many issues that need to be addressed before this promise can be realized. Perhaps foremost among the issues associated with macromolecular treatments is the efficient delivery of the therapeutic molecules to the site(s) in the body where they are needed. The ideal delivery vehicle would be one that could be systemically administered and then home to tumor cells wherever they occur in the body. A variety of delivery systems (“vectors”) have been tried, including viruses and liposomes. The infectivity that makes viruses attractive as delivery vectors also poses their greatest drawback. Residual viral elements can be immunogenic, cytopathic or recombinogenic. The generation of novel viruses with new targets for infection also raises the theoretical possibility that, once introduced into patients, these viruses could be transformed via genetic alteration into new human pathogens. Consequently, a significant amount of attention has been directed at non-viral vectors for the delivery of molecular therapeutics. The liposome approach offers a number of advantages over viral methodologies for gene delivery. Most significantly, they lack immunogenicity. Moreover, since liposomes are not infectious agents capable of self-replication, they pose no risk of evolving into new classes of infectious human pathogens.
Targeting cancer cells via liposomes can be achieved by modifying the liposomes so that they selectively deliver their payload to tumor cells. Surface molecules can be used to target liposomes to tumor cells, because the molecules that decorate the exterior of tumor cells differ from those on normal cells. For example, if a liposome has the protein transferrin (Tf) or an antibody that recognizes transferrin receptor (TfR) on its surface, it will home to cancer cells that have higher levels of the TfR. Such liposomes designed to home to tumors have been likened to “smart” bombs capable of seeking out their target.
Failure to respond to therapy represents an unmet medical need in the treatment of many types of cancer, including prostate cancer. Often when cancer recurs, the tumors have acquired increased resistance to radiation or chemotherapeutic agents. The incorporation into currently used cancer therapies of a new component which results in radio-/chemo-sensitization would have immense clinical relevance. One way in which such sensitization could be achieved is via gene therapy (i.e., delivery of a gene the expression of which results in increased sensitization). In PCT patent application WO 00/50008 (published 31 Aug. 2000), incorporated herein by reference, we provided proof-of-principle that an anti-transferrin receptor single chain antibody (TfRscFv) can be chemically conjugated to a cationic liposome. Moreover, this TfRscFv directed liposome delivery system can deliver genes and other molecules systemically and specifically to tumors.
Immunoliposomes and Cationic Polymers as Gene Transfer Vehicles
As noted above, some of the problems associated with using viral vectors could be circumvented by non-viral gene transfer vectors. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems (31, 32). Cationic liposomes are composed of positively charged lipid bilayers and can be complexed to negatively charged, naked DNA by simple mixing of lipids and DNA such that the resulting complex has a net positive charge. The complex can be bound and taken up by cells in culture with moderately good transfection efficiency (33). Features of cationic liposomes that make them versatile and attractive for DNA delivery include: simplicity of preparation; the ability to complex large amounts of DNA; versatility in use with any type and size of DNA or RNA; the ability to transfect many different types of cells, including non-dividing cells; and lack of immunogenicity or biohazardous activity (reviewed in 34, 35). More importantly from the perspective of human cancer therapy, cationic liposomes have been proven to be safe and efficient for in vivo gene delivery (33, 34, 36). At least 99 clinical trials have been approved using cationic liposomes for gene delivery (37), and liposomes for delivery of small molecule therapeutics (e.g., antifungal agents) are already on the market.
Researchers also have considered the suitability of cationic polymers as transfer vectors for delivery of therapeutic agents in vivo. For example, Polyethyleneimine (PEI) is the organic macromolecule with the highest cationic-charge-density potential, and a versatile vector for gene and oligonucleotide transfer in vitro and in vivo, as first reported by Boussif et al. (66). Since then, there has been a flurry of research aimed at this polycation and its role in gene therapy (73). Cell-binding ligands can be introduced to the polycation to 1) target specific cell types and 2) enhance intracellular uptake after binding the target cell (13). Erbacher et al. (67) conjugated the integrin-binding peptide 9-mer RGD via a disulfide bridge and showed physical properties of interest for systemic gene delivery.
The transfection efficiency of both cationic liposomes and cationic polymers, such as PEI, can be increased dramatically when they bear a ligand recognized by a cell surface receptor. Receptor-mediated endocytosis represents a highly efficient internalization pathway present in eukaryotic cells (38, 39). The presence of a ligand on a liposome facilitates the entry of DNA into cells through initial binding of ligand by its receptor on the cell surface followed by internalization of the bound complex. Transferrin receptor (TfR) levels are elevated in various types of cancer cells including, but not limited to, breast, pancreatic, head and neck, and prostate cancers (40), even those prostate cell lines derived from human lymph node and bone metastases (40-43). Elevated TfR levels also correlate with the aggressive or proliferative ability of tumor cells (44). Therefore, TfR is a potential target for drug delivery in the therapy of malignant cell growth (45, 46). In our laboratory, we have prepared transferrin-complexed cationic liposomes with tumor cell transfection efficiencies in SCCHN of 60%-70%, as compared to only 5-20% by cationic liposomes without ligand (47). Also see published PCT patent application WO 00/50008.
In addition to the use of ligands that are recognized by receptors on tumor cells, specific antibodies also can be attached to the liposome surface (48) enabling them to be directed to specific tumor surface antigens (including but not limited to receptors) (49). These “immunoliposomes,” especially the sterically stabilized immunoliposomes, can deliver therapeutic drugs to a specific target cell population (50). Parks et al. (51) found that anti-HER-2 monoclonal antibody (MAb) Fab fragments conjugated to liposomes could bind specifically to a breast cancer cell line, SK-BR-3, that overexpresses HER-2. The immunoliposomes were found to be internalized efficiently by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Moreover, the anchoring of anti-HER-2 Fab fragments enhanced their inhibitory effects. More recently, Park et al. (23) used an anti-HER-2 immunoliposome composed of long circulating liposomes chemically conjugated to anti-HER-2 monoclonal antibody scFv fragments to deliver doxorubicin to breast cancer tumors even though HER-2 was not over-expressed. A number of other studies have been published which have employed antibodies against tumor specific antigens coupled to liposomes, primarily sterically stabilized liposomes, to target tumor cells for delivery of prodrugs and drugs in vitro or in vivo (52-56). These studies demonstrated the utility of immunoliposomes for tumor-targeting drug delivery. The combination of cationic liposome-gene transfer and immunoliposome techniques appears to be a promising system for targeted gene therapy and is the subject of this application.
Progress in biotechnology has allowed the derivation of specific recognition domains from MAb (57). The recombination of the variable regions of heavy and light chains and their integration into a single polypeptide provides the possibility of employing single-chain antibody derivatives (designated scFv) for targeting purposes. Thus, a scFv based on the anti-TfR MAb 5E9 (52) contains the complete antibody binding site for the epitope of the TfR recognized by this MAb as a single polypeptide chain of approximate molecular weight 26,000. This TfRscFv is formed by connecting the component VH and VL variable domains from the heavy and light chains, respectively, with an appropriately designed peptide. The peptide bridges the C-terminus of the first variable region and N-terminus of the second, ordered as either VH-peptide-VL or VL-peptide-VH. The binding site of an scFv can replicate both the affinity and specificity of its parent antibody combining site.
The TfRscFv has advantages in human use over the Tf molecule itself or even an entire MAb to target liposomes or cationic polymers to cancer cells with elevated levels of the TfR for a number of reasons. First, the size of the scFv (˜28 kDa) is much smaller than that of the Tf molecule (˜80 kDa) or the parental MAb (˜150 kDa). The scFv-liposome-therapeutic agent complex or scFv-polymer-therapeutic agent complex thus may exhibit better penetration into small capillaries characteristic of solid tumors. Second, the smaller scFv also has practical advantages related to its production as a recombinant protein. Large scale production of the TfRscFv will be required for the therapy envisioned in this invention to be taken into eventual human trials. Third, the scFv is a recombinant molecule (not a blood product like Tf) and, therefore, presents no issues related to potential contamination with blood borne pathogens. Additional advantages of using the TfRscFv relate to the fact that Tf interacts with the TfR with high affinity only after the ligand is loaded with iron. Large-scale production of liposomes containing iron-loaded Tf may present practical challenges. Thus, use of TfRscFv enables the tumor cell TfR to be targeted by a liposomal therapeutic complex that does not contain iron (itself implicated in cancer (58)). Fourth, without the Fc region of the MAb, the problem of non-antigen-specific binding through Fc receptors is eliminated (57).
p53 Tumor Suppressor Gene and the Pathogenesis of Prostate Cancer
The tumor suppressor gene p53 plays a crucial role in diverse cellular pathways including those activated in response to DNA damage, such as DNA repair, regulation of the cell cycle and programmed cell death (apoptosis) (1). Malfunctions of these critical cell pathways are associated with the process of tumorigenesis. Loss of functional p53, which has been implicated in over 60% of human cancers, can occur either through mutations in the p53 gene itself (the most common occurrence), or through other mechanisms such as amplification of the MDM-2 gene (found in certain sarcomas, and other cancers), or association of p53 with the E6 protein of human papilloma virus (which likely plays a role in cervical carcinoma) (2).
The loss of p53 function is of relevance to a broad array of cancer types, with non-functional p53 associated with, for example, 15-50% of breast cancer, 25-70% of metastatic prostate cancer, 25-75% of lung cancer, and 33-100% of head and neck cancers (3). The presence of mutant p53 also has been associated with an unfavorable prognosis for many human cancers including lung, colon, and breast (3), and mutant p53 is rarely found in some of the most curable forms of cancer e.g., Wilm's tumor, retinoblastoma, testicular cancer, neuroblastoma and acute lymphoblastic leukemia (4). In addition, p53 protein transcriptionally regulates genes involved in angiogenesis, a process required for solid tumor growth (5). Volpert et al. have proposed that development of the angiogenic phenotype for these tumors requires the loss of both p53 alleles (6).
Since it appears that most anti-cancer agents work by inducing apoptosis (20), inhibition of or changes in this pathway may lead to failure of therapeutic regimens. A direct link has been suggested between mutations in p53 and resistance to cytotoxic cancer treatments (both chemo- and radiotherapy (21)). It has also been suggested that the loss of p53 function may contribute to the cross-resistance to anti-cancer agents observed in some tumor cells (22).
Restoration of p53 function could, therefore result in sensitization of primary prostate tumors and even metastases to radio-/chemo-therapy. The introduction of wtp53 has been reported to suppress, both in vitro and in mouse xenograft models, the growth of various types of malignancies, e.g., prostate (23,24), head and neck (25,26), colon (27), cervical (28) and lung (15,29) tumor cells. However, p53 alone, while being able to partially inhibit tumor growth, has not been shown to be able to eliminate established tumors. Significantly, however, we have demonstrated that the combination of systemically delivered liposome-p53 and radiation led to complete long-term tumor regression of established head and neck xenograft tumors (25,30).
In summary, the implication of the p53 gene in a significant fraction of human cancers makes it one of the premiere candidates for cancer gene therapy. Based on a growing body of evidence related to p53 functions, effective restoration of these functions in tumor cells might be expected to re-establish normal cell growth control, restore appropriate responses to DNA-damaging agents (e.g., chemotherapy and radiotherapy), and to impede angiogenesis.
The sensitization of tumors to chemotherapy and radiation could lower the effective dose of both types of anticancer modalities, correspondingly lessening the severe side effects often associated with these treatments. Until now the vast majority of p53 gene therapy protocols have employed wtp53 gene replacement alone. Based upon the current literature and our data (30, 59), it appears that wtp53 replacement alone, while able to inhibit tumor growth to some extent, is insufficient to eliminate tumors long term. Therefore, it appears that a combinatorial approach involving both standard therapy and targeted gene therapy has substantial promise as a novel and more effective clinical modality for cancer treatment. Moreover, the demonstrated tumor cell selectivity of our systemically delivered ligand-liposome wtp53 complex indicates the potential of this method to sensitize even the distant micrometastases that are the ultimate cause of so many prostate cancer deaths.
Components of intracellular signaling pathways including, but not limited to receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (non-RTKs), are crucial mediators of many critical pathways including cell proliferation, differentiation, migration, angiogenesis, cell cycle regulation etc. (Baselga, Science 312, 1175-1178 (2006), Arora and Scholar, J Pharmacol Exp Ther 315, 971-979 (2005), Krause and Van Etten N Eng J Med 353, 172-187 (2005)). Many of these crucial pathways are deregulated in cancer cells. Thus, RTKs and non-RTKs are good targets for cancer therapeutics. One class of such therapeutics are small molecules including, but not limited to those that target growth factor receptors and thus affect these signaling pathways (Imai and Takoka, Nature Reviews: Cancer 6, 714-727 (2006)). These inhibitors compete with ATP (ATP mimetics) and inhibit kinase activity. One of the first successful small molecule inhibitors is Imatinib mesylate (GLEEVEC®). This small molecule inactivates the kinase activity of BCR-ABL fusion protein in CML (Druker Trends in Molecular Medicine 8, S14-S18 (2002)), and has shown significant efficacy in the treatment of patients with Philadelphia chromosome positive CML. It is also an inhibitor of other TKs, including KIT and PDGFRα and PDGFRβ KIT is involved in metastatic GISTs and the two platelet derived growth factor receptors are involved in tumors such as glioblastoma and dermatofibrosarcoma protuberans. Because it is a member of the EGF superfamily, EGFR is also a logical target for small molecule inhibitors. Gefitinib (IRESSA®) (Herbst et al Nature reviews Cancer 4, 9560965 (2004)) and erlotinib (TARCEVA®) (Minna and Dowell Nature Reviews Drug Discovery Suppl. S14-S15 (2005)) selectively inhibit EGFR and have shown efficacy against EGFR expressing cancers such as NSCLC and squamous cell carcinomas of the head and neck. They have also shown efficacy in Phase II trials in combination with chemotherapeutic agents. The combination of erlotinib and chemotherapeutic agent gemcitibine (an anti-metabolite) has been approved for use in treating advanced pancreatic cancer. Several Phase III trials of Gefitinib are on going (Chai and Grandis Current Treat Opin Oncol 7, 3-11 (2006)).
Small Molecule agents can translocate through the plasma membrane and interact with the cytoplasmic domain of the cell surface receptors and intracellular signaling molecules. Thus, small molecules are also being developed that affect cancer cell proliferation and survival by inhibiting RAS prenylation, RAF-MEK kinase, PI3Kinase, the mTOR pathway (the mammalian target of rapamycin, and even heat shock protein 9). They can also affect cell adhesion and invasion by inhibiting SRC kinase or matrix metalloproteinases. Inhibition of vascular endothelial growth factor (VEGF) by small molecules can also inhibit neovascularization of tumors.
A new type of small molecule agent, Sorafenib (Nexavar) exerts its inhibitory effect on different isoforms of RAF serine kinase as well as various RTKs (VEGF, EGFR, and PDGF) (Arora and Scholar, J Pharmacol Exp Ther 315, 971-979 (2005)). This “dual-action” kinase inhibitor shows broad-spectrum anti-tumor activity by inhibiting tumor proliferation and angiogenesis (Marx Science 308, 1248-1249 (2005)). Sunitinib malate (SUTENT®) is also a multitargeted TK inhibitor of VEGF, PDGFR, KIT and FLIT3 (Marx Science 308, 1248-1249 (2005)). Potential targets for small molecule agents have also been identified in the ubiquitin-proteosome pathway which is crucial in cell cycle arrest and apoptosis (programmed cell death). A selective, reversible inhibitor of the chymotryptic protease in the 26S proteosome, Bortezomb (Velcade), has been reported to be effective against various cancers, particularly hematological malignancies
The anti-EGFR TK inhibitors are synthetic chemicals of ˜500 Da that are administered orally, with half-lives of ˜46 hours for (IRESSA®) and ˜36 hours for TARCEVA®. Because they are administered orally rather than intravenously, plasma concentrations at the same dose of the small molecule therapeutic can vary between patients (Dancy and Sausville Nature Rev Drug Discov 2, 116-124 (2003)). This is a disadvantage of these agents as currently used. Thus encapsulating small molecule agents in a tumor-targeting delivery complex that can be administered intravenously consistently at the same dose, such as that of this invention, would improve their use as therapeutic agents. Furthermore, encapsulation in such a ligand-liposome complex would protect the small molecule agents from degradation further enhancing their efficacy. Untargeted orally administered small molecule agents are not specific for tumor cells, a fact which increases the risk of normal cell toxicity and adverse side effects. While these side effects are generally mild (e.g. rash, acne, dry skin and pruritis) the gastrointestinal toxities (nausea, vomiting, anorexia and particularly diarrhea) can be dose limiting. The most common side effect, skin rash, is possibly due to non-specific effects on the target kinase in the epidermis (Herbst et al Clin Lung Caner 4, 366-369 (2003)). Thus, delivery by a tumor cell specific agent could decrease this problem. The most severe toxicity reported to date is with IESSA® (gefitinib): interstitial pneumonitis, a form of pneumonia characterized by non-infectious inflammation and fibrosis in the lower respiratory tract. Over 170 patients have died of this disease after treatment with IRESSA® (Arora and Scholar, J Pharmacol Exp Ther 315, 971-979 (2005)). Recently, chest CT and radiographic imaging has shown that gefitinib-related interstitial lung disease is similar to that of pulmonary damage caused by conventional antineoplastic agents and there may be a direct cytotoxic effect. Therefore, the use of a tumor cell specific agent to deliver IRESSA®, or any small molecule agent, directly to the tumor cells might provide a solution to the problem of interstitial pneumonitis and other side effects. Moreover, direct delivery to the site where needed (primary and metastatic disease), including in the brain, by the method of this invention would also result in a decrease in the dose required for effective treatment, a further benefit currently not possible.